GSM7049772: A549CC_4C_rep2; Homo sapiens; OTHER
4C-seq was performed as previously described using EcoR I for the first digestion, and Csp6 I for the second digestion. In brief, cells were cross-linked in 2% formaldehyde for 10 min at room temperature, glycine (V900144, Sigma) was added to a final concentration of 0.2 M to quench the reaction. After crosslinking, cells were rinsed with cold PBS at room temperature twice and incubated in lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% CA630, 0.1% Triton X-100 and protease inhibitor cocktail (04693132001, Roche)) while tumbling for 1 h at 4 ℃. Cells were then pelleted, washed, and resuspended in EcoR I buffer. SDS was added to a final concentration of 0.3% for 1 h in a thermomixer at 37 °C and 900 rpm. Triton X-100 was then added to a final concentration of 1.8% and incubated in a thermomixer at 37 °C and 900 rpm for 3 h. 200 units EcoR I enzyme (New England Biolabs, R3101M) were added for a 4 h incubation in a thermomixer at 37 °C and 900 rpm; another 200 units EcoR I were added for overnight incubation. EcoR I was heat inactivated by incubating at 65 °C for 20 min. Cells were suspended in ligation buffer while 4,000 units of T4 ligase (New England Biolabs, M0202M) were added and incubated more than 8 h at 16 °C and 300 rpm. After ligation, 300 μg Proteinase K were added for overnight incubation at 65 °C and 400 rpm. DNA was purified and digested with 90 units Csp6 I (New England Biolabs, R0639S) and incubated for 5 h in a thermomixer at 25 °C and 500 rpm, followed by heat inactivation of enzyme at 65 °C for 20 min. DNA were then diluted with ligation buffer and 100 units T4 ligase were added for ligation overnight at 16 °C and 300 rpm. The second ligation circularized DNA were purified and amplified using bait-specific inverse primers (29 bp downstream of the rs1663689 and 55bp downstream of the EcoR I site to identify the allele at SNP and unknown DNA sequences interacting with the bait). Primers were extended with dangling Illumina adaptor sequences. After amplification, PCR products were pooled and purified. Sequencing reads were demultiplexed and trimmed of the primer sequence using sabre tool (https://github.com/najoshi/sabre), mapped to the human reference genome (hg38) with Bowtie2 (version 2.2.1) and converted to restriction fragment space. Interactions were called on single 4C experiments with peakC, using a sliding window of 21 fragments. Libraries were prepared according to Illumina's instructions accompanying the Paired-End DNA Samples Prep Kit. Briefly, DNA fragment end repair, add A base to 3'end, and connect sequencing adapter. PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 2500 following the manufacturer's protocols.